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Foot Ulcers: Characterising the Microbiome of Ulcers Karen Smith1, Andrew Collier2, Eleanor M

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Foot Ulcers: Characterising the Microbiome of Ulcers Karen Smith1, Andrew Collier2, Eleanor M Smith et al. BMC Microbiology (2016) 16:54 DOI 10.1186/s12866-016-0665-z RESEARCH ARTICLE Open Access One step closer to understanding the role of bacteria in diabetic foot ulcers: characterising the microbiome of ulcers Karen Smith1, Andrew Collier2, Eleanor M. Townsend1,3, Lindsay E. O’Donnell3, Abhijit M. Bal2, John Butcher1, William G. Mackay1, Gordon Ramage3* and Craig Williams1 Abstract Background: The aim of this study was to characterise the microbiome of new and recurrent diabetic foot ulcers using 16S amplicon sequencing (16S AS), allowing the identification of a wider range of bacterial species that may be important in the development of chronicity in these debilitating wounds. Twenty patients not receiving antibiotics for the past three months were selected, with swabs taken from each individual for culture and 16S AS. DNA was isolated using a combination of bead beating and kit extraction. Samples were sequenced on the Illumina Hiseq 2500 platform. Results: Conventional laboratory culture showed positive growth from only 55 % of the patients, whereas 16S AS was positive for 75 % of the patients (41 unique genera, representing 82 different operational taxonomic units (OTU’s). S. aureus was isolated in 72 % of culture-positive samples, whereas the most commonly detected bacteria in all ulcers were Peptoniphilus spp., Anaerococcus spp. and Corynebacterium spp., with the addition of Staphylococcus spp. in new ulcers. The majority of OTU’s residing in both new and recurrent ulcers (over 67 %) were identified as facultative or strict anaerobic Gram-positive organisms. Principal component analysis (PCA) showed no difference in clustering between the two groups (new and recurrent ulcers). Conclusions: The abundance of anaerobic bacteria has important implications for treatment as it suggests that the microbiome of each ulcer “starts afresh” and that, although diverse, are not distinctly different from one another with respect to new or recurrent ulcers. Therefore, when considering antibiotic therapy the duration of current ulceration may be a more important consideration than a history of healed ulcer. Keywords: Diabetes,Diabeticfootulcer,Infection,Microbiome, Next generation sequencing, 16S amplicon sequencing Background high plantar pressures [2]. Diabetic foot ulcers have a A serious complication of diabetes is the development of significant negative impact on health and quality of life foot ulcers. Patients with diabetes are believed to have a and are the most common cause of hospitalisation in pa- 12–25 % lifetime risk of developing a foot ulcer [1]. The tients with diabetes [3]. aetiology of diabetic foot ulceration is complex. Foot ul- Diabetic foot ulcers often heal very slowly, due to cers often develop due to a combination of intrinsic fac- diabetes-associated micro-vascular disease and impaired tors, such as peripheral neuropathy, poor extremity host immune response, and these open wounds provide perfusion, foot deformity, changes to the plantar foot a niche for infection [4, 5]. Bacteria can exist within the soft tissues plus extrinsic mechanical factors, such as wound as multi-layered microbial communities, known as biofilms, surrounded by a self-produced protective * Correspondence: [email protected] extracellular ‘slime’ [6]. The presence of a biofilm makes 3 Infection and Immunity Research Group, Glasgow Dental School, School of infections very difficult to resolve, as the structure Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK shields the encased cells from antimicrobial agents and Full list of author information is available at the end of the article © 2016 Smith et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Smith et al. BMC Microbiology (2016) 16:54 Page 2 of 12 the host immune system, allowing bacteria to persist Table 1 Clinical laboratory culture of diabetic foot wound and impair healing [7]. Many foot ulcers fail to heal and samples cause serious complications, such as osteomyelitis. Infec- Sample number New or recurrent ulcer Bacteria isolated tion is the most common cause of lower limb amputa- 1 Recurrent NSGa tion in diabetic foot ulcers [3]. In the UK, more than 2 Recurrent NSG one hundred amputations are carried out each week in 3 Recurrent Candida spp. individuals with diabetes [1]. In addition to the signifi- cant trauma, the cost to the NHS of treating infected 4 New Mixed growth + anaerobe diabetic foot ulcers and resulting amputations is esti- 5 New Mixed growth + anaerobe mated to be in the region of £900 million per year [8]. 6 Recurrent S. aureus + anaerobe Standard treatment of diabetic foot ulcers involves de- 7 New S. aureus + anaerobe bridement of the necrotic tissue, management of infection 8 New NSG and off-loading of the ulcer [9]. Infection is routinely con- 9 New S. aureus + beta-haemolytic firmed by laboratory culture of bacteria in a swab taken Streptococcus (Group G) from the wound. Culture-dependent methods show bias 10 Recurrent NSG towards microorganisms that are able to grow well on la- 11 New NSG boratory culture media. More fastidious organisms may not be identified, resulting in a delay in the appropriate 12 New S. aureus treatment [10, 11]. Molecular methods are advancing and 13 New S. aureus + beta-haemolytic becoming more accessible and affordable (including 16S Streptococcus (Group G) amplicon sequencing [16S AS]), and it is now possible to 14 Recurrent S. aureus use bacterial DNA from the wound site to identify the 15 Recurrent NSG pathogens present [12]. A greater understanding of both 16 Recurrent NSG the bacteria present in diabetic foot ulcers and how these 17 New S. aureus bacteria interact with one another, and the host, will be 18 New NSG crucial for the development of reliable models of infection and effective treatments. 19 Recurrent S. aureus It was our hypothesis that the specific microbiome as- 20 Recurrent NSG sociated with new and recurrent diabetic foot ulcers dif- aNSG - No significant growth fered, and that this impacted the ability to effectively manage these wounds. Therefore, the aim of this study aureus was confirmed in 50 % of the samples tested by was to use 16S AS technologies to characterise the PCR. Spearman’s rank correlation showed that this cor- microbiome of new and recurrent ulcers and to under- related to culture results (p < 0.05, CI 0.5761–0.9642). take comparative analyses to gain understanding of whether the presence of certain microorganisms were Microbiome analysis diabetic foot ulcers by 16S amplicon associated with the inability of an ulcer to heal. sequencing DNA was successfully amplified and sequenced from 16 of the Results samples (75 %), including 9 from new ulcers and 7 from recur- The detection of bacteria in diabetic foot ulcers by rent ulcers. These 16 samples produced an average of 1,767,142 conventional laboratory culture and molecular methods sequence reads, which were filtered for quality and assigned an By conventional laboratory culture, 11 samples (55 %) OTU using a minimum sequence similarity of 97 %. The abun- were positive for microbial growth by standard aerobic dance of each species in wound samples of greater than 0.5 % and anaerobic culture methods (4 recurrent ulcer sam- was reported. Raw data sets are available from the following ples and 7 new ulcer samples) (Table 1). The remaining website: http://www.ncbi.nlm.nih.gov/bioproject/304940. 9 swab samples (45 %) produced no significant growth In the 16 ulcer samples there were 41 unique genera, (6 recurrent and 3 new ulcer samples). In the samples representing 82 different OTU’s, which ranged from 21 that were positive for bacterial growth, 6 contained more different species (sample 4) to only 2 species (sample 9). than one species. S. aureus was isolated in 8 of culture- The species identified in new and non-healing ulcers are positive samples, anaerobes were isolated from 4 sam- displayed in Table 2, along with their frequency of detec- ples, beta-haemolytic streptococci from 2 samples, and tion. In new ulcers there were 94 different OTU’s Candida spp. was identified alone in only 1 sample. One identified; most frequently detected genera were Pep- control swab sample collected from the healthy skin toniphilus (6 samples), Staphylococcus (5 samples), (sample 19) showed a moderate growth of S. aureus. Anaerococcus (5 samples) and Corynebacterium (4 samples). These results are summarised in Table 1. Moreover, S. In recurrent ulcers 73 unique OTU’s were identified; Smith et al. BMC Microbiology (2016) 16:54 Page 3 of 12 Table 2 List of species identified in the new and recurrent ulcers by 16S AS Species identified No. of samples Gram type Oxygen tolerance New Recurrent Actinobaculum massiliense 1 0 Gram Positive Facultative anaerobe Actinobaculum schaalii 2 2 Gram Positive Facultative anaerobe Actinomyces europaeus 0 1 Gram Positive Facultative anaerobe Actinomyces hominis 0 1 Gram Positive Facultative anaerobe Actinomyces neuii 0 1 Gram Positive Facultative
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